The present invention relates to a test medium and method for the detection, quantification, identification and/or differentiation of biological materials in a sample which may contain a plurality of different biological materials.
Bacteria are the causative factor in many diseases of humans, higher animals and plants, and are commonly transmitted by carriers such as water, beverages, food and other organisms. The testing of these potential carriers of bacteria is of critical importance and generally relies on “indicator organisms.” Borrego et al., Microbiol. Sem. 13:413-426, (1998). For example, Escherichia coli (E. coli) is a gram negative member of the family Enterobacteriaceae which is part of the normal intestinal flora of warm blooded animals, and its presence indicates fecal contamination (e.g., raw sewage). Even though most strains of E. coli are not the actual cause of disease, their presence is a strong indication of the possible presence of pathogens associated with intestinal disease, such as cholera, dysentery, and hepatitis, among others. Consequently, E. coli has become a prime indicator organism for fecal contamination, and as a result, any method which differentiates and identifies E. coli from other bacteria is very useful.
Others members of the family Enterobacteriaceae, commonly referred to as “general coliforms,” especially the genera Citrobacter, Enterobacter and Klebsiella, are also considered to be significant indicator organisms for the quality of water, beverages and foods. Therefore, tests to identify and differentiate general coliforms from E. coli are also very useful. Also, various species of the genus Aeromonas have been shown to not only be potential pathogens, but to have a correlation to other indicator organisms (Pettibone et al., J. Appl. Microbiol. 85:723-730 (1998)). Current test methods to identify, separate and enumerate Aeromonas spp. from the very similar Enterobacteriaceae have been lacking and most of the current methods utilizing enzyme substrates do not separate Aeromonas spp. from Enterobacteriaceae due to their almost identical biochemical profiles. Any method that depends upon the identification of general coliforms by means of a β-galactosidase substrate either does not differentiate Aeromonas spp. from general coliforms or eliminates Aeromonas from the sample by the use of specific inhibitors (antibiotic such as cefsulodin). Brenner et al., Appl. Envir. Microbio. 59:3534-44 (1993). They do not differentiate, identify and enumerate Aeromonas along with E. coli and general coliforms. Landre et al., Letters Appl. Microbiol. 26:352-354(1998). Improved test methods to effectively identify, separate and enumerate such bacterial types are needed, and there is a continuing search for faster, more accurate, easier to use and more versatile test methods and apparatus in this area.
Numerous test methods have been utilized to determine, identify and enumerate one or more indicator organisms. Some of these test methods only indicate the presence or absence of the microorganism, while others also attempt to quantify one or more of the particular organisms in the test sample. For example, a qualitative test referred to as the Presence/Absence (or P/A) test, may be utilized to determine the presence or absence of coliforms and E. coli in a test sample. A test medium including the β-galactosidase substrate O-nitrophenyl-β-D-galactopyranoside (ONPG), and the β-glucuronidase substrate 4-methyl-umbrelliferyl-β-D-glucuronide (MUG), is inoculated with the test sample. To differentiate the general coliforms from E. coli, this test relies on the fact that generally all coliforms produce β-galactosidase, whereas only E. coli also produces β-glucuronidase in addition to β-galactosidase. If any coliforms are present (including E. coli), the broth medium turns a yellow color due to the activity of the galactosidase enzyme on the ONPG material, causing the release of a diffusible yellow pigment. If E. coli is present, the broth medium will demonstrate a blue fluorescence when irradiated with ultraviolet rays, due to the breakdown of the MUG reagent with the release of the fluorogenic dye caused by the production of the glucuronidase enzyme. These reactions are very specific, and allow the presence of both coliforms in general, as well as E. coli to be identified in a single sample. A disadvantage of this test is that it is not directly quantitative for either bacterial type, since both reagents produce diffusible pigments. A second disadvantage is that there may a false positive coliform reaction if Aeromonas spp. are present in the test sample. This has been shown to be possible even when there are inhibitors present to supposedly prevent this from occurring (Landre et al., Letters Appl. Microbiol. 26:352-354 (1998)). The test also requires specific equipment for producing the ultraviolet rays. Further, this test may only be used to detect coliforms and E. coli. Other important microorganisms, such as the strain E. coli 0157 which is glucuronidase negative, are not detected, nor are other non-galactosidase-glucuronidase producing microorganisms.
The Violet Red Bile Agar (VRBA) method has been used to determine the quantity of both coliform and E. coli in a test sample. The test medium used in this method includes bile salts (to inhibit non-coliforms), lactose and the pH indicator neutral red. As coliforms (including E. coli) grow in the medium, the lactose is fermented with acid production, and the neutral red in the area of the bacterial colony becomes a brick red color. The results of this test are not always easy to interpret, and in order to determine the presence of E. coli, confirming follow-up tests, such as brilliant green lactose broth fermentation, growth in EC broth at 44.5° C. and streaking on Eosin Methylene Blue Agar (EMBA), must be performed.
The Membrane Filter (MF) method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. This method is used most often when bacterial populations are very small, and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated, and the bacterial colonies growing on the membrane filter surface are counted and evaluated. This method is widely used and provides good results when combined with proper reagents and media. A disadvantage of this method is that it is expensive and time-consuming. It also does not work well with solid samples, or samples with high particulate counts. The MF method can be used in conjunction with the inventive method described in this application.
The m-Endo method is also used to determine the quantity of E. coli and general coliforms and is an official USEPA approved method for testing water quality. The medium is commonly used with a membrane filter and E. coli and general coliform colony forming units (CFU) grow as dark colonies with a golden green metallic sheen. Due to a proven high rate of false positive error, typical colonies must be confirmed by additional testing. Standard Methods for the Examination of water and Wastewater, 20th Edition, 9-10 &9-60 (1998).
Other tests, such as the Most Probable Number (MPN), utilize lactose containing broths (LST, BGLB, EC) to estimate numbers of general coliforms and E. coli, but have also been shown to have high rates or error as well as being cumbersome and slow to produce results. Evans et al., Appl. Envir. Microbiol. 41:130-138 (1981).
The reagent 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) is a known test compound for identifying coliforms. When acted on by the β-galactosidase enzyme produced by coliforms, X-gal forms an insoluble indigo blue precipitate. X-gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. X-gal has the advantage over the compound ONPG, described above, in that it forms a water insoluble precipitate rather than a diffusible compound, thereby enabling a quantitative determination of coliforms to be made when the test sample is incorporated into or onto a solidified medium, or when coliform colonies grow on the surface of a membrane filter resting on a pad saturated with a liquid medium or on a membrane filter resting on a solid medium. Further, it does not require the use of ultraviolet light.
A similar compound, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) is a known test compound for identifying E. coli. When acted on by the β-glucuronidase enzyme produced by most E. coli, X-gluc forms an insoluble indigo blue precipitate. X-gluc has the advantage over the compound MUG, described above, in that it forms a water insoluble precipitate, rather than a diffusible compound, thereby enabling a quantitative determination of E. coli to be made when the test sample is incorporated into or onto a solidified medium. X-gluc and its ability to identify E. coli are described in Watkins, et al., Appl. Envir. Microbiol. 54:1874-1875 (1988). A similar compound, indoxyl-β-D-glucuronide, which also produces sharp blue colonies of E. coli, was described in Leg, et al., Can. J. Microbiol. 34:690-693 (1987).
Although X-gal and X-gluc are each separately useful in the quantitative determination of either coliforms (X-gal) or E. coli (X-gluc), these indicator compounds have the disadvantage that they each contain the same chromogenic component. Therefore, they cannot be used together to identify and distinguish both E. coli and general coliforms in a single test with a single sample, since they both generate identically hued indigo blue colonies. A person using both reagents together would be able to quantitatively identify the total number of coliforms, the same as if X-gal were used alone, but would not be able to indicate which of the colonies were E. coli and which were other coliforms besides E. coli. 
A recently developed and highly commercially successful test method and test medium for quantitatively identifying and differentiating general coliforms and E. coli in a test sample is described in U.S. Pat. Nos. 5,210,022, and 5,393,662, both of which share common inventorship with the present application and which are hereby incorporated by reference. This method and test medium improves upon prior art methods by allowing the quantitative identification of general coliforms and E. coli in a single sample. Additional confirmatory tests are not necessary. The test sample is added to a medium containing a β-galactosidase substrate, such as 6-chloroindolyl-β-D-galactoside, and a β-glucuronidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc). The β-galactosidase substrate is capable of forming a water insoluble precipitate of a first color upon reacting with β-galactosidase, and the β-glucuronidase substrate is capable of forming a water insoluble precipitate of a second color, contrasting with the first color, upon reacting with β-glucuronidase. As a result, general coliforms may be quantified by enumerating the colonies of the first color (having β-galactosidase activity), and E. coli may be quantified by enumerating the colonies of the second color (having both β-galactosidase and β-glucuronidase activity). This technology has been widely copied.
Another recently developed test method and apparatus provides excellent results for the differentiation and identification of general coliforms, E. coli and E. coli 0157 strains and non-coliform Enterobacteriaceae. The method and test medium are described in U.S. Pat. No. 5,726,031, which shares common inventorship with the present application, and which is hereby incorporated by reference.
A certain class of substrates, referred to herein as “nonchromogenic,” have been used to detect various microorganisms. A dipslide for detecting E. coli using hydroxy-quinoline-β-D-glucuronide is disclosed by Dalet et al., J. Clin. Microbiol, 33(5):1395-8 (1995). Similarly, a technique for detection of E. coli in an agar-based medium using 8-hydroxyquinoline-β-D-glucuronide is disclosed by James et al., Zentralbl Bakteriol Mikrobiol Hyg [A], 267(3):316-21 (1988).
It is desirable to further improve the distinguishing colors generated in the test media. That is to say, in prior art test media which detect and distinguishing two biological entities, confusion may arise between the two colors which show in the media.
Further, it is desirable to be able to identify and differentiate other closely related organisms, such as members of the families Aeromonaceae, Vibrionaceae, and Salmonella. For example, the genus Aeromonas is closely related to coliforms and gives an almost identical biochemical test pattern. Further, the genus Vibrio is also an important type of bacteria that grows under the same general conditions as coliforms. It is known to distinguish Aeromonas colonies from general coliforms by testing all colonies in a given sample for the presence of cytochrome oxidase. Undesirably, however, this requires another set of tests. Further, Aeromonas is common in water and food, and it is commonly indicated in test samples as general coliforms, which results in high a false positive error for general coliforms by most current test methods. The Aeromonas can be prevented from interfering with the coliform results by adding certain antibiotics to the medium. However, the antibiotic amounts added must be carefully controlled. Further, the antibiotics which have been conventionally used have short life spans in the media so that they lose their potency quickly in other than a frozen condition. It may often be desirable to be able to culture, identify and enumerate Aeromonas spp. which cannot be done if they are inhibited.
Further, in those cases where it is desirable to inhibit Aeromonas, it is desirable for a better method of so doing, one in which the shelf life of the medium is not appreciably reduced by the inclusion of an inhibitor.
Additionally, it is also desirable to distinguish strains of Salmonella from E. coli, general coliforms and Aeromonas. 